This invention claims priority of a German patent application DE 100 03 570.1 which is incorporated by reference herein.
The invention concerns a microscope assemblage, in particular for confocal scanning microscopy, having a light source for illuminating a specimen to be examined and at least one fluorescent-light detector for the detection of fluorescent light generated in the specimen and at least one transmitted-light detector for the detection of transmitted light passing through the specimen.
Microscope assemblages of the kind cited above are known from practical use and exist in a wide variety of embodiments. One example of such a microscope assemblage is constituted by a confocal scanning microscope in which a specimen to be examined is scanned with a light beam. The microscope generally comprises a light source and a focusing optical system with which the light of the source is focused onto an aperture stop. A beam splitter, a scanning device for beam control, a microscope optical system, a detection stop, and detectors for the detection of detected or fluorescent light are provided.
The illuminating light is usually coupled in via the beam splitter. The focus of the light beam is moved with the scanning device in one specimen plane. This is usually done with two mirrors which are tilted, the deflection axes usually being located perpendicular to one another, so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, with the aid of galvanometer positioning elements. In this xe2x80x9cdescanxe2x80x9d arrangement that is usual, the fluorescent or reflected light coming from the specimen arrives via the same scanning mirror back at the beam splitter and passes through it, then being focused onto the detection stop behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection stop, so that a point datum is obtained and yields, by scanning of the specimen, a three-dimensional image. Illumination and detection occur here on the objective side, i.e. by way of the microscope optical system.
In a transmitted-light arrangement it is also possible, for example, to detect the fluorescent light or the transmitted light (the transmitted exciting light) on the condenser side, i.e. on the side of a condenser arranged after the specimen. The detected light beam then does not pass via the scanning mirror to the detector. An arrangement of this kind is called a xe2x80x9cnon-descanxe2x80x9d arrangement.
In the transmitted-light arrangement, a condenser-side detection stop would be necessary for detection of the fluorescent light in order, as in the case of the descan arrangement described, to achieve three-dimensional resolution. In the case of two-photon excitation, however, a condenser-side detection stop can be dispensed with, since the excitation probability depends on the square of the photon density or intensity, which of course is much greater at the focus than in the adjacent regions. A very large proportion of the fluorescent light to be detected therefore derives, with high probability, from the focus region, rendering superfluous any further differentiation, using a stop arrangement, between fluorescent photons from the focus region and fluorescent photons from the adjacent regions.
Especially given that the yield of fluorescent photons with two-photon excitation is in any case low, a non-descan arrangement, in which less light is generally lost along the detected light path, is of interest But even when fluorescent light is observed in this manner, cell outlines, for example, cannot be detected sufficiently well because they are not labeled in living preparations, so that it would be desirable simultaneously to be able to observe the transmitted light, which would allow definite conclusions to be drawn.
Microscope assemblages do exist in which objective-side fluorescence detection on the one hand, and condenser-side transmission detection on the other hand, are possible. In this context, however, changing from objective-side fluorescence detection to condenser-side transmission detection and vice versa requires a mechanical switching operation in which mirrors and filters must be mechanically displaced. This can cause shocks to the specimen which destroy it. Experiments with micropipette arrangements, in particular, are thus almost ruled out.
The present invention is therefore based on the object of describing a microscope assemblage of the kind cited initially in which a very wide variety of experiments, in particular experiments with micropipette arrangements, can reliably be made with a high level of detection in each case.
The aforesaid object is achieved by a microscope assemblage which comprises: a light source for illuminating a specimen, at least one fluorescent-light detector for the detection of fluorescent light generated in the specimen and at least one transmitted-light detector for the detection of transmitted light passing through the specimen, wherein the fluorescent-light and transmitted-light detectors are arranged to enable simultaneous detection of fluorescent and transmitted light.
The object is achieved as well by a microscope assemblage which comprises: a light source for illuminating a specimen, at least one fluorescent-light detector for the detection of fluorescent light generated in the specimen; at least one transmitted-light detector for the detection of transmitted light passing through the specimen, wherein the fluorescent-light and transmitted-light detectors are arranged to enable simultaneous detection of fluorescent and transmitted light and a first polarization device is provided between the light source and the specimen, a second polarization device in provided after the specimen.
Furthermore, the object is achieved by an other embodiment of the microscope assemblage, which comprises: a light source for illuminating a specimen, at least one fluorescent-light detector for the detection of fluorescent light generated in the specimen, wherein the specimen defines a top side facing the light source and a bottom side facing away from the light source, at least one transmitted-light detector for the detection of transmitted light passing through the specimen, and an additional light source being arranged on the side of the specimen facing away from the light source.
What has been recognized according to the present invention is that by skillful arrangement of the fluorescent-light and transmitted-light detectors, the aforesaid object is achieved in surprisingly simple fashion. For this purpose, the fluorescent-light and transmitted-light detectors are arranged in such a way that simultaneous detection of fluorescent light and transmitted light is made possible. Switching over between transmitted-light and fluorescent-light detection is no longer necessary, so that mechanical shocks to the sample or specimen are prevented. A high level of detection is thus achieved in terms of both the detection of transmitted light and the detection of fluorescent light, even with sensitive specimens.
The microscope assemblage according to the present invention consequently provides a microscope assemblage with which a very wide variety of experiments, in particular experiments with micropipette arrangements, can reliably be made with a high level of detection in each case.